Genetic structure and diversity analysis of the primary gene pool of chickpea using SSR markers.

Members of the primary gene pool of the chickpea, including 38 accessions of Cicer arietinum, six of C. reticulatum and four of C. echinospermum grown in India were investigated using 100 SSR markers to analyze their genetic structure, diversity and relationships. We found considerable diversity, with a mean of 4.8 alleles per locus (ranging from 2 to 11); polymorphic information content ranged from 0.040 to 0.803, with a mean of 0.536. Most of the diversity was confined to the wild species, which had higher values of polymorphic information content, gene diversity and heterozygosity than the cultivated species, suggesting a narrow genetic base for cultivated chickpea. An unrooted neighbor-joining tree, principal coordinate analysis and population structure analysis revealed differentiation between the cultivated accessions and the wild species; three cultivated accessions were in an intermediate position, demonstrating introgression within the cultivated group. Better understanding of the structure, diversity and relationships within and among the members of this primary gene pool will contribute to more efficient identification, conservation and utilization of chickpea germplasm for allele mining, association genetics, mapping and cloning gene(s) and applied breeding to widen the genetic base of this cultivated species, for the development of elite lines with superior yield and improved adaptation to diverse environments.

Simultaneous measurements of ossicular velocity and intracochlear pressure leading to the cochlear input impedance in gerbil.

Recent measurements of three-dimensional stapes motion in gerbil indicated that the piston component of stapes motion was the primary contributor to intracochlear pressure. In order to make a detailed correlation between stapes piston motion and intracochlear pressure behind the stapes, simultaneous pressure and motion measurements were undertaken. We found that the scala vestibuli pressure followed the piston component of the stapes velocity with high fidelity, reinforcing our previous finding that the piston motion of the stapes was the main stimulus to the cochlea. The present data allowed us to calculate cochlear input impedance and power flow into the cochlea. Both the amplitude and phase of the impedance were quite flat with frequency from 3 kHz to at least 30 kHz, with a phase that was primarily resistive. With constant stimulus pressure in the ear canal the intracochlear pressure at the stapes has been previously shown to be approximately flat with frequency through a wide range, and coupling that result with the present findings indicates that the power that flows into the cochlea is quite flat from about 3 to 30 kHz. The observed wide-band intracochlear pressure and power flow are consistent with the wide-band audiogram of the gerbil.

Scala vestibuli pressure and three-dimensional stapes velocity measured in direct succession in gerbil.

It was shown that the mode of vibration of the stapes has a predominant piston component but rotations producing tilt of the footplate are also present. Tilt and piston components vary with frequency. Separately it was shown that the pressure gain between ear canal and scala vestibuli was a remarkably flat and smooth function of frequency. Is tilt functional contributing to the pressure in the scala vestibuli and helping in smoothing the pressure gain? In experiments on gerbil the pressure in the scala vestibuli directly behind the footplate was measured while recording simultaneously the pressure produced by the sound source in the ear canal. Successively the three-dimensional motion of the stapes was measured in the same animal. Combining the vibration measurements with an anatomical shape measurement from a micro-CT (CT: computed tomography) scan the piston-like motion and the tilt of the footplate was calculated and correlated to the corresponding scala vestibuli pressure curves. No evidence was found for the hypothesis that dips in the piston velocity are filled by peaks in tilt in a systematic way to produce a smooth middle ear pressure gain function. The present data allowed calculations of the individual cochlear input impedances.

The response of the apical turn of cochlea modeled with a tuned amplifier with negative feedback.

In an earlier study [Hear. Res. 149 (2000) 55] velocity amplitudes of the outer Hensen's cell (HC) and basilar membrane (BM) were measured before, and at different times, after, sacrificing the animal. The velocity amplitude changed in a way that was characteristic of a negative feedback amplifier. A simple negative feedback amplifier model was proposed to explain the magnitude of the HC and BM velocity changes at CF. In the experiment tuning changed as well, both at the HC and BM. The model has now been extended to include tuning changes. The model response is compared with the experimental observations. The model is able to account quantitatively for the following experimental observations: (i) At the HC the tuning broadens and velocity decreases slowly after sacrifice. (ii) At the BM tuning sharpens and velocity increases at a faster rate. (iii) The velocity increase at BM is much larger than the decrease at HC.

Non-linear response to amplitude-modulated waves in the apical turn of the guinea pig cochlea.

Mechanical vibrations of the Hensen's cells were measured in the apical turn of the cochlea in living guinea pigs, in response to amplitude-modulated (AM) sound. The FFT of the input wave consisted of spectral components at the carrier frequency C and two sidebands (C+/-M) separated from the carrier by the modulation frequency M. The FFT of the velocity response consisted of components at: (i) the modulation frequency M, and harmonics n M; (ii) Carrier frequency C and sidebands (C+/-n M); (iii) harmonics of the carrier frequency and their side bands (2C+/-n M); (3C+/-n M); (4C+/-n M); em leader n=1,2,3, em leader,10. The carrier and the first pair of side bands were broadly tuned and nearly linear. Other components were sharply tuned and highly non-linear, suggesting a different origin. Evidence is presented that these components are generated in the non-linear stereocilia dynamics. An important function of this non-linearity is to demodulate the AM wave to extract information contained in the modulation.

Amplification in the apical turn of the cochlea with negative feedback.

The apical turn of the anesthetized guinea pig cochlea was opened to examine the basilar membrane optically through the intact Reissner's membrane. Vibrations of the outer Hensen's cell and the basilar membrane (BM) adjacent to and about 130 microm below the level of the Hensen's cell were measured. Outer Hensen's cell vibration at the characteristic frequency was up to 900 times higher compared to the BM amplitude. After sacrifice BM vibration increased while Hensen's cell vibration decreased. The magnitude and sequence of change after sacrifice can best be explained by the presence of negative feedback between reticular lamina and BM. In other experiments using ototoxic drugs that damage outer hair cells, similar changes in Hensen's cell and BM vibration were observed. These results show that the apical turn behavior is different from that observed by other investigators in the basal turn. The potential benefits of the negative feedback are discussed. The presence of negative feedback would explain the linearity at the fundamental frequency observed in the apical turn of cochlea.

Mechanical nonlinearity in the apical turn of the guinea pig organ of Corti.

Confocal microscopy was used to view the sealed apical turn of the cochlea in a living guinea pig, and to identify the cochlear structures through the intact Reissner's membrane. X, Y and Z coordinates for each point of interest were recorded. A confocal laser heterodyne interferometer measured the cellular vibration in response to acoustical signals applied to the ear. Velocity time waveforms were recorded at 32 frequencies between 25 and 2500 Hz at each point of measurement. To characterize the vibration pattern of the organ of Corti, vibrations at multiple locations along a radial track of the reticular lamina were measured before and after sacrificing the animal. Amplitude and phase tuning curves of the fundamental and the second harmonic, velocity time waveforms, and FFTs of time waveforms are compared before and after sacrifice. The results show that a sharply tuned nonlinear part of the response disappears shortly after sacrifice.

Vibrations of the guinea pig organ of Corti in the apical turn.

Vibrations of the organ of Corti were measured in response to sound applied to the ear in the apical turn of a living guinea pig. Measurements were made at 29 points on the Reissner's membrane (RM) at 10 micro spacing along a radial track. Measurements also included 22 points on the reticular lamina (RL), Claudius' cells and osseous spiral lamina. Our goal was to characterize the vibration of the RM and the RL with high spatial resolution along a radial axis. The tuning and spatial patterns of the RM are compared in the radial direction with those for the RL at the fundamental frequency and at the second harmonic. The shape of the RM tuning curve changes with radial position, and differ significantly from those observed at the RL. These results support our earlier findings (Hao and Khanna, Hear. Res. 99 (1996) 176-189).

Nonlinearity in the apical turn of living guinea pig cochlea.

Mechanical vibrations were measured at the apical turn in living guinea pig cochlea, in response to sinusoidal acoustic stimuli, using heterodyne interferometry. The cochlea was sealed and the vibrations were measured at different cellular locations along a radial track at the level of reticular lamina and one point on the osseous spiral lamina. Averaged time waveforms were recorded at each test frequency. The nonlinearity in the apical turn is demonstrated by the distortion in the time waveforms and the richness of the harmonic components in their Fourier transforms. Tuning curves and input/output curves for the fundamental and harmonics components are shown. The fundamental component is essentially linear below about 90 dB SPL while the harmonics display strong nonlinearity and saturation. Negative feedback in the apical turn of the cochlea linearizes the response at the fundamental frequency.

Micromechanical effects in the cochlea of tetracaine.

Local anesthetics applied in the tympanic cavity have earlier been shown to affect the gross receptor potentials in reducing the cochlear microphonics and increasing the positive summating potential. To study the effects of this drug on the mechanical responses in the cochlea, vibrations were measured using laser heterodyne interferometry in an isolated in vitro temporal bone preparation from the guinea pig. Measurements were made at a set of frequencies in the fourth cochlear turn from the Hensen's cells and the outer hair cells in response to sound applied to the ear. The tuning curves of the fundamental and the second harmonic components of the vibratory responses were plotted. When 2 mM tetracaine was applied, the high frequency slope of the second harmonic curve shifted down in frequency, this caused the frequency of the maximum of second harmonic tuning to shift down. These changes were reversible when tetracaine was washed out. Observations were also made in the temporal bone preparation in vitro with a confocal microscope. Fluorescent probes were used to label various structures in the organ of Corti. Optical sections were obtained by tilting the organ permitting a view from the side like a radial section through the organ. Images were acquired before, during and after application of tetracaine and were later analyzed with a computer program. Simultaneously, cochlear microphonics and the summating potential were obtained to monitor the electrical response of the preparation. Although the cochlear microphonics and summating potential decreased when 2 mM tetracaine was applied, structural changes were not measurable in the organ of Corti. The decrease was reversible when tetracaine was washed out. It is concluded that tetracaine affected the high frequency part of the non-linear second harmonic component, possibly by lowering the stiffness of the stereocilia bundle or the body of the outer hair cells.

Reticular lamina vibrations in the apical turn of a living guinea pig cochlea.

The reticular lamina of the apical turn of a living guinea pig cochlea was viewed through the intact Reissner's membrane using a slit confocal microscope. Vibrations were measured at selected identified locations with a confocal heterodyne interferometer, in response to tones applied with an acoustic transducer coupled to the ear canal. The position coordinates of each location were recorded. Mechanical tuning curves were measured along a radial track at Hensen's cells, outer hair cells, inner hair cells and at the osseous spiral lamina, over a frequency range of 3 kHz, using five sound pressure levels (100, 90, 80, 70 and 60 dB SPL). The carrier to noise ratio obtained throughout the experiments was high. The response shape at any measuring location was not found to change appreciably with signal level. The response shape also did not change significantly with the radial position on the reticular lamina. However, the response magnitude increased progressively from the inner hair cell to the Hensen's cell. The observed linearity of response at the fundamental frequency is explained by the presence of negative feed back in the apical turn of the cochlea.

Vibration of reflective beads placed on the basilar membrane.

Most investigators place reflective beads on the basilar membrane to measure its vibration with optical methods. It is therefore important to find out if the beads faithfully follow the motion of the structures on which they are placed. Vibration of the beads on the basilar membrane and basilar membrane adjacent to the beads are measured in the third turn of the guinea pig cochlea in a temporal bone preparation. It is shown that the beads do not follow the motion of the organ. The mechanism by which this departure may occur is investigated by modeling the motion of the beads on the Claudius' cells.

Mechanical response characteristics of the hearing organ in the low-frequency regions of the cochlea.

1. With the use of an in vitro preparation of the guinea pig temporal bone, in which the apical turns of the cochlea are exposed, the mechanical and electrical responses of the cochlea in the low-frequency regions were studied during sound stimulation. 2. The mechanical characteristics were investigated in the fourth and third turns of the cochlea with the use of laser heterodyne interferometry, which allows the vibratory responses of both sensory and supporting cells to be recorded. The electrical responses, which can be maintained for several hours, were recorded only in the most apical turn. 3. In the most apical turn, the frequency locations and shapes of the mechanical and electrical responses were very similar. 4. The shapes of the tuning curves and the spatial locations of the frequency maxima in the temporal bone preparation compared very favorably with published results from in vivo recordings of hair cell receptor potentials and sound-induced vibrations of the Reissner's membrane. 5. Compressive nonlinearities were present in both the mechanical and the electrical responses at moderate sound pressure levels. 6. The mechanical tuning changed along the length of the cochlea, the center frequencies in the fourth and third turns being approximately 280 and 570 Hz, respectively. 7. The mechanical responses of sensory and supporting cells were almost identical in shape but differed significantly in amplitude radially across the reticular lamina.

Acoustically induced vibrations of the Reissner's membrane in the guinea-pig inner ear.

In the inner ear, the Reissner's membrane separates the scala vestibuli from the scala media and is thus of importance for maintaining a positive endocochlear potential. The motion of the membrane is thought to be driven by the vibrations of the underlying hearing organ caused by a hydromechanical coupling between the structures. Since the Reissner's membrane is relatively easily accessible in the cochlea its vibratory response has been used as a measure of the micromechanical behaviour of the hearing organ. To determine whether this indirect measure revealed the true characteristics of the hearing organ, experiments were performed using laser heterodyne interferometry in an in vitro preparation of the guinea-pig temporal bone. Interferometric measurements at the Reissner's membrane and at the surface of the hearing organ directly beneath made it possible to compare the mechanical tuning characteristics of both structures. It was found that the mechanical response characteristics of the Reissner's membrane differed considerably from the hearing organ. The tuning frequency was different and only minor changes in the maximal vibration amplitude were seen when measuring at different radial locations. However, the shape of the response curve changes with location. The Reissner's membrane response appeared to be affected by the mechanical vibrations originating both at the middle ear ossicles and at the hearing organ. It is concluded that the Reissner's membrane response is a poor indicator of cochlear mechanics and that investigations of cochlear micromechanics should be performed directly at the level of the hearing organ.

Reissner's membrane vibrations in the apical turn of a living guinea pig cochlea.

Mechanical tuning curves were recorded at several radial locations on the Reissner's membrane, over a wide range of frequencies, and sound pressure levels. The position coordinates of each location were also recorded. The shape of the tuning curves changed dramatically with the radial location. Near the outer edge of the cochlea the response was broadly tuned, with a maxima near 300 Hz, while near the inner edge the response showed at least three maxima and minima. Responses were also measured at the reticular lamina. The shapes of the frequency responses at the Reissner's membrane are quite different from those measured at the reticular lamina below it.

The vibration pattern of the hearing organ in the waltzing guinea-pig measured using laser heterodyne interferometry.

The mechanical tuning characteristics of the hearing organ were measured in response to sound stimulation using laser heterodyne interferometry in in vitro preparations of temporal bones from waltzing guinea-pigs expressing different degrees of hearing organ and sensory cell degeneration. Measurements were made at various stages of structural changes allowing us to correlate structure and mechanical function. It was found that the characteristic frequency of the response at a given location in the cochlea occurred at lower frequencies than what is normally seen and that the sharpness of the mechanical tuning was considerably reduced when sensory hair cells were absent and the hearing organ structurally altered. However, even when extensive hair cell degeneration was evident a residual mechanical tuning was present. These results further support the concept that the sensory hair cells plays a key role in determining normal auditory tuning characteristics. It is suggested that the basilar membrane mechanics gives rise to a broadly tuned mechanical response on which a sharper tuning mechanism, originating from the hair cells, is superimposed.

Investigating routes to chaos in the guinea-pig cochlea using the continuous wavelet transform and the short-time Fourier transform.

The continuous wavelet transform (CWT) and the short-time Fourier transform (STFT) were used to analyze the time course of cellular motion in the guinea pig inner ear. The velocity responses of individual outer hair cells and Hensen's cells to amplitude modulated (AM) acoustical signals applied to the ear canal displayed characteristics typical of nonlinear systems, such as the generation of spectral components at harmonics of the carrier frequency. Nonlinear effects were particularly pronounced at the highest stimulus levels, where half-harmonic (and sometimes quarter-harmonic) components were also seen. The generation of these components was consistent with the behavior of a dynamical system entering chaos via a period-doubling route. A negative-stiffness Duffing oscillator model yielded period-doubling behavior similar to that of the experimental data. We compared the effectiveness of the CWT and the STFT for analyzing the responses to AM stimuli. The CWT (calculated using a high-Q Morlet-wavelet basis) and the STFT were both useful for identifying the various spectral components present in the AM velocity response of the cell. The high-Q Morlet wavelet CWT was particularly effective in distinguishing the lowest frequency components present in the response, since its frequency resolution is appreciably better than the STFT at low frequencies. Octave-band-based CWTs (using low-Q Morlet, Meyer, and Daubechies 4-tap wavelets) were largely ineffective in analyzing these signals, inasmuch as the frequency spacing between neighboring spectral components was far less than one octave.

Shearing motion in the hearing organ measured by confocal laser heterodyne interferometry.

To investigate the presence of the postulated shearing motion in the micromechanics of the inner ear during sound stimulations we measured the vibratory response of the tectorial membrane and the reticular lamina in the third cochlear turn in an isolated temporal bone preparation using confocal laser heterodyne interferometry. The mechanical response of the tectorial membrane had the same frequency of maxima as the underlying reticular lamina, but was not as sharply tuned. When the two-dimensional motion was calculated from measurements made from several viewing angles it was found that the vibration of the reticular lamina had significant components both normal and tangential to its surface. The tectorial membrane motion, however, was primarily in a direction approximately perpendicular to the surface of the reticular lamina. The results indicate that shearing motion is produced predominantly by the radial motion of the reticular lamina.

Spatial distribution of sound pressure and energy flow in the ear canals of cats.

Spatial pressure distributions have been measured in the ear canals of ten cats and analyzed to obtain the energy reflection properties of the middle ear over a 10- to 25-kHz range of frequencies. Considerable intersubject variability is observed, much of which can be correlated with the condition of the tympanic membrane. For ears judged to be in good condition, reflection coefficients typically take values of about 0.2 between 15 and 25 kHz, indicating good matching of the dynamical properties of the auditory system to the ear canal sound field. At lower frequencies, the reflection coefficients tend to be somewhat higher and at higher frequencies the reflection coefficients increase quite rapidly with frequency. For ears judged to be in poorer condition, energy reflection coefficients of 0.5 and 0.9 were determined for the 15- to 25-kHz range. The variations of sound pressure along the canal (about 10 dB, even in well-coupled systems) confirm that single point pressure measurements may be inappropriate for defining the acoustical input at higher frequencies and new measures for specifying the input should be investigated. The net flow of acoustic energy into the auditory system, the sound power, is one possibility. Some initial measurements of sound power, obtained from analysis of the spatial pressure distributions, are presented.

A method for determining three-dimensional vibration in the ear.

In the classical concept of the middle ear function the malleus rotates around a fixed axis which implies that at small amplitudes of vibration its displacement is essentially one dimensional. As a consequence malleus vibrations have been measured previously along a single viewing axis. As a first step in the study of the complete malleus motion we determined the three dimensional components at a single point (umbo) of the manubrium. To define 3-D motion it is in principle necessary to measure the vibrations from widely different observation angles. The viewing angles are limited however in our case by the ear canal geometry to about +/-15 degrees. In order to resolve the 3-D components under these conditions it is necessary to measure the vibration components with high accuracy. Amplitude and phase of the umbo vibrations were measured with a heterodyne interferometer over a wide frequency range (100 Hz to 20 kHz). The system included a two axis goniometer with the axes of rotation positioned at the focal plane of the interferometer objective lens. It was therefore possible to change the viewing angle in small increments around two orthogonal axes while keeping the same point in focus. From a redundant set of measurements the three orthogonal components of vibration were calculated by least squares fitting. The vector sum of the three components gives the three dimensional motion of the observed point. The vibration of the point on the umbo was found not to follow a straight line but an elliptical path instead. The shape of the ellipse and the inclination of the plane of the ellipse with respect to the stationary malleus position changed with frequency. These observations are consistent with our earlier findings that the mode of malleus vibration changes with frequency [Decraemer et al. (1991) Hear. Res. 54, 305-318].